The aim of the work described in this proposal is to test various aspects of mechanism of metal ion catalysis of amide hydrolysis in an effort to clarify possible roles of the metal in metallopeptidases. In preliminary model studies, large rate enhancements by metal ions have been observed for amides bearing aliphatic amine leaving groups. This observation requires that the metal ion is greatly accelerating the normally rate- limiting breakdown of the tetrahedral intermediate (TI), but the factors responsible for this are not evident at this time, and most previous workers have focused on the effect of the metal on the TI formation step. A full characterization of the kinetically significant rate terms operating in these model reactions together with studies on additional models designed to limit the mode of interaction between the metal ion and the amide moiety is planned. The complexation of metal ions to the model substrates will be studied by a combination of spectroscopy and potentiometric titration. Kinetic studies will be performed using either pH stat, colorimetric, or aliquot methods, and will be conducted as a function of pH, metal ion identity and concentration, and temperature. In addition to the theoretical importance to biochemistry, information revealed by these studies may be useful in the design of specific inhibitors and new prodrug strategies based on the hydrolytic action of metallopeptidases.